SUMMARY In recent years, it has become increasingly clear that the material properties of ribonucleoprotein (RNP) granules, which are formed via liquid-liquid phase separation, play crucial roles in both cellular physiology and pathology. Nevertheless, mechanistic understandings of the molecular determinants and modulators of RNP granule viscoelastic phases remain incomplete due to the limitations of currently available techniques to probe for protein condensate dynamics across single-molecule to mesoscale. The goal of this proposal is to address this critical gap by the development of a multi-parametric experimental toolbox that simultaneously reports on RNP condensate structure and dynamics across different length-scales, with high sensitivity. Our approach will feature correlative multicolor single-molecule fluorescence microscopy, dual-trap optical tweezers, and microfluidics. Utilizing our novel toolbox, we will decipher the mechanisms of liquid-to-liquid and liquid-to-solid phase transitions of intracellular RNP condensates, processes that critically contribute to the onset or development of many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Commonly used fluorescence microscopy techniques, such as fluorescence recovery after photobleaching (FRAP), offer only probe-specific protein/RNA diffusivity within the RNP granules. In contrast, our proposed correlative force-fluorescence microscopy platform will provide a multiscale view of RNP condensate dynamics by taking advantage of optical tweezer-based rheological and fluid dynamics measurements in conjunction with quantification of protein dynamics using single-molecule fluorescence. We hypothesize that (a) a hierarchy of protein-protein and protein-nucleic acid interactions determines both nanoscale RNP dynamics and micron-scale material properties of the condensate, and (b) post-translational modifications, RNA/DNA and ligand binding, and pathologic mutations modulate the material properties of RNP condensates by manipulating the long-range and short-range inter-molecular forces. Overall, our research program will address three Key Challenges (KCs): (a) we will develop a novel experimental toolbox based on correlative multi-color confocal fluorescence microscopy and dual-trap optical tweezer that simultaneously reports on molecular and mesoscale protein condensate structure and dynamics (KC 1), (b) we will apply our toolbox to map the transition pathways of physiologic RNP granules to pathologic states in c9orf72 repeat expansion disorder (KC 2), and (c) we will identify mechanisms of ligand-dependent transcriptional condensate regulation at DNA enhancer sites (KC 3). Our studies will provide new insights into the determinants of functional RNP condensate material states, dynamics, and composition, as well as identify novel pathways of these granules? pathologic alterations.